As a technique of measuring and analyzing a target analyte contained in a liquid sample such as blood, plasma, and urine, a liquid sample analyzing method and a liquid sample analyzing apparatus have been known in which a liquid sample is added to a test piece called a biosensor, the liquid sample is extended on an extended layer on the test piece by using chromatography, a target analyte immobilized at a predetermined point is read using the optical property of the target analyte, and the target analyte in the liquid sample is analyzed (for example, patent documents 1 and 2).
As shown in FIG. 5(a), a test piece 20 is configured such that an extended layer 3 containing a porous carrier is provided on a support 2 shaped like a rectangular thin plate, an added portion (so-called dropped portion) 4 for receiving a liquid sample is provided on one end of the test piece 20, and the liquid sample added to the added portion 4 is extended over the extended layer 3 by capillarity. On the extended layer 3, a labeled reagent portion 5 containing a labeled reagent is provided near the added portion and an immobilized reagent portion 6 containing an immobilized specific antibody is provided at a predetermined point. When a liquid sample such as blood and plasma is added to the added portion 4, the liquid sample is extended over the extended layer 3 on the test piece 20 while reacting with the labeled reagent of the labeled reagent portion 5, the labeled reagent having being bound by a specific antibody (different from the specific antibody of the immobilized reagent portion 6). Further, the liquid sample undergoes a combination reaction with the specific antibody of the immobilized reagent portion 6, so that the immobilized reagent portion 6 is colored according to the concentration of a target analyte in the liquid sample. After that, the liquid sample analyzing apparatus measures the coloring of the immobilized reagent portion 6 of a test piece 1, converts the coloring into the concentration of the target analyte, and outputs the analysis result.
As shown in FIG. 6, a liquid sample analyzing apparatus 10′ includes: a holder 11 that holds the test piece 20 placed on the holder 11; a light source 12 that emits light to the test piece 20 and is made up of, e.g., a lamp, a light-emitting diode, or a semiconductor laser; a diaphragm 13 that limits scattered light (transmitted light or reflected light) from the light source 12; a condenser lens 14 that focuses light having passed through the diaphragm 13; an image element 15 such as a CCD on which the light is focused to form an image of the surface of the test piece 20; and a control unit (not shown) that converts an electric signal from the image element 15 into a digital signal, performs processing such as image processing to calculate as an absorbance the degree of coloring (brightness) of the immobilized reagent portion 6 serving as a colored portion on the test piece 20, calculates the concentration of a target analyte in a liquid sample by using the absorbance according to a stored concentration conversion formula, and outputs the concentration. Advantageously, since the image element 15 such as a CCD is used, it is possible to remove a colored portion in the background of the extended layer 3 of the test piece 20 by a subtraction and increase the accuracy of measurement by correcting the coloring through color tone correction.
FIG. 20 shows a measurement method using a test piece according to the prior art.
Reference numeral 1 denotes the test piece containing the immobilized reagent portion 6 that is colored according to the concentration of a measurement object in a liquid sample. An optical system for reading the degree of coloring of the immobilized reagent portion 6 is made up of the light source 12, the diaphragm 13, the condenser lens 14, and the image element 15.
The test piece 1 is irradiated with light from the light source 12, and the light scattered on the surface of the test piece 1 is received by the image element 15 through the diaphragm 13 and the condenser lens 14.
The step of processing an image captured by the image element 15 includes image generating step S1, coloring degree determining step S2, and result output step S3.
In the image generating step S1, an image signal outputted from the image element 15 is stored as a gray image in an image memory (not shown). In the coloring degree determining step S2, the degree of coloring of the immobilized reagent portion 6 is calculated. In the result output step S3, the measurement result is outputted to a display device (not shown) such as a liquid crystal display.
On this test piece 20, when both sides of the extended layer 3 of the test piece 20 are opened, the liquid sample and the labeled regent that are extended over the extended layer 3 by capillarity may flow or evaporate out of both sides of the extended layer 3. Thus a large quantity of liquid sample may be necessary or the penetration direction may vary and cause unstable coloring. In order to address this problem, patent document 3 proposes a configuration in which both sides of a test piece are sealed by melting and hardening with laser light, the sides being parallel to the extending direction (penetration direction) of the test piece.
With this configuration, both sides of the test piece are sealed to prevent a liquid sample and a labeled reagent from evaporating or flowing out of both sides of the test piece. Thus it is possible to reduce the quantities of the liquid sample and the labeled reagent and improve the accuracy by flow adjustment such that the liquid sample and the labeled reagent are properly extended. As shown in FIG. 5(b), the extended layer 3 may be partially melted and hardened by emitting laser light to a portion disposed inside both sides of a test piece 20′ in the width direction of the test piece 20′, that is, by emitting laser light along lines extended in the longitudinal direction of the test piece 20′ along both sides of the test piece 20′, so that a passage region 3a where the liquid sample flows on the extended layer 3 is smaller in width than the test piece 20′. In FIG. 5(b), reference numeral 7 denotes overflow blocking lines for preventing the liquid sample and the labeled reagent from extending and flowing out of both sides of the test piece 20′ (in the width direction of the test piece 20′). The overflow blocking lines 7 are formed by melting and hardening with laser light. Reference character 3a denotes the passage region of the extended layer 3. The liquid sample and the labeled reagent actually pass through the passage region. Reference character 3b denotes non-passage regions where the liquid sample and the labeled reagent do not flow on the extended layer 3. In FIG. 5(b), the two overflow blocking lines 7 are formed at each of two points arranged in the width direction of the extended layer 3.
In this configuration, the overflow blocking lines 7 substantially parallel to both sides of the test piece 20′ are formed inside both sides of the test piece 20′, thereby substantially reducing the passage width of the extended layer 3 on which the liquid sample and the labeled reagent are extended. This configuration can reduce the required quantity of the liquid sample. Further, the immobilized reagent portion 6 serving as a colored portion and the labeled reagent portion 5 are reduced in width according to the passage width. Thus it is possible to reduce the quantities of the immobilized reagent and the labeled reagent.
As has been discussed, the overflow blocking lines 7 are formed on the test piece 20′ such that the passage width of the extended layer 3 on which the liquid sample and the labeled reagent are extended is substantially smaller than the width of the test piece 20′. In this case, coloring occurs only at points disposed inside the overflow blocking lines 7 (passage region 3a) in the width direction of the test piece. Thus in the case of the test piece 20′ on which the overflow blocking lines 7 are formed thus, when the degree of coloring is measured on the immobilized reagent portion 6 serving as a colored portion, it is preferable that the width of the passage region 3a substantially acting as the extended layer 3 is recognized beforehand and a portion where the immobilized reagent portion 6 is provided is set as a measured region.
However, actually because of various factors reducing positioning accuracy, e.g., a displacement of the test piece 20′ fixed on the holder 11 and displacements of the immobilized reagent portion 6, the extended layer 3, and the overflow blocking lines 7 on the test piece 20′ in a manufacturing process, it cannot be assumed that the passage region 3a and the immobilized reagent portion 6 are correctly placed at the same positions in each measurement. Therefore, in each measurement, it is preferable to recognize the test piece 20′ through the image element 15 and detect the passage width of the extended layer 3.
In a conventional method of recognizing the passage width of the extended layer 3, an image of the extended layer 3 is generated in a state in which the liquid sample is added to the test piece 20′ and is extended over the extended layer 3. Further, a pixel is detected on a scanning line L1 crossing the extended layer 3 containing the overflow blocking lines 7. FIG. 7 shows that the test piece 20′ (see FIG. 5(b)) on which the overflow blocking lines 7 are formed is measured by the liquid sample analyzing apparatus 10′. FIG. 8(a) shows an example of an image. FIG. 8(b) shows brightness at a point corresponding to the image. It is determined that a region from the center of the extended layer 3 in the width direction to small fluctuations in brightness, for example, a region D of FIG. 8(b) is located inside the overflow blocking lines 7.    Patent document 1: Japanese Patent Laid-Open No. 7-5110    Patent document 2: Japanese Patent Laid-Open No. 2001-4613    Patent document 3: Japanese Patent No. 3542999    Patent document 4: Japanese Patent Laid-Open No. 2000-266752    Patent document 5: International Publication No. WO 01/092884